Culture-Independent Analysis of Lactic Acid Bacteria Diversity Associated with Mezcal Fermentation

Mezcal is an alcoholic beverage obtained from the distillation of fermented juices of cooked Agave spp. plant stalks (agave must), and each region in Mexico with denomination of origin uses defined Agave species to prepare mezcal with unique organoleptic characteristics. During fermentation to produce mezcal in the state of Tamaulipas, not only alcohol-producing yeasts are involved, but also a lactic acid bacterial community that has not been characterized yet. In order to address this lack of knowledge on this traditional Mexican beverage, we performed a DGGE-16S rRNA analysis of the lactic acid bacterial diversity and metabolite accumulation during the fermentation of a typical agave must that is rustically produced in San Carlos County (Tamaulipas, Mexico). The analysis of metabolite production indicated a short but important malolactic fermentation stage not previously described for mezcal. The denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes showed a distinctive lactic acid bacterial community composed mainly of Pediococcus parvulus, Lactobacillus brevis, Lactobacillus composti, Lactobacillus parabuchneri, and Lactobacillus plantarum. Some atypical genera such as Weissella and Bacillus were also found in the residual must. Our results suggest that the lactic acid bacteria could strongly be implicated in the organoleptic attributes of this traditional Mexican distilled beverage.     

Diversity of Lactic Acid Bacteria Associated with Fresh Coffee Cherries in Taiwan

A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800 m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200 m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substance-producing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.     

Core Fluxome and Metafluxome of Lactic Acid Bacteria under Simulated Cocoa Pulp Fermentation Conditions

In the present work, simulated cocoa fermentation was investigated at the level of metabolic pathway fluxes (fluxome) of lactic acid bacteria (LAB), which are typically found in the microbial consortium known to convert nutrients from the cocoa pulp into organic acids. A comprehensive ¹³C labeling approach allowed to quantify carbon fluxes during simulated cocoa fermentation by (i) parallel ¹³C studies with [¹³C₆]glucose, [1,2-¹³C₂]glucose, and [¹³C₆]fructose, respectively, (ii) gas chromatography-mass spectrometry (GC/MS) analysis of secreted acetate and lactate, (iii) stoichiometric profiling, and (iv) isotopomer modeling for flux calculation. The study of several strains of L. fermentum and L. plantarum revealed major differences in their fluxes. The L. fermentum strains channeled only a small amount (4 to 6%) of fructose into central metabolism, i.e., the phosphoketolase pathway, whereas only L. fermentum NCC 575 used fructose to form mannitol. In contrast, L. plantarum strains exhibited a high glycolytic flux. All strains differed in acetate flux, which originated from fractions of citrate (25 to 80%) and corresponding amounts of glucose and fructose. Subsequent, metafluxome studies with consortia of different L. fermentum and L. plantarum strains indicated a dominant (96%) contribution of L. fermentum NCC 575 to the overall flux in the microbial community, a scenario that was not observed for the other strains. This highlights the idea that individual LAB strains vary in their metabolic contribution to the overall fermentation process and opens up new routes toward streamlined starter cultures. L. fermentum NCC 575 might be one candidate due to its superior performance in flux activity.     

产黏乳酸菌的发酵特性研究

通过对2株产黏乳酸球菌的发酵特性比较研究,以感观、酸度、活菌数对数值、培养时间、粘度及乳清析出率为考察指标,结果表明:乳酸球菌Q26具备较好的产黏、产香特性,遗传性质较稳定.通过与乳酸杆菌G18进行发酵应用试验,确定G18和Q26存在共生关系,混合发酵的酸牛乳乳清析出率为0.1％,粘度为4 528mPa·s.通过保质期试验,产品在28、20、4℃下保质期分别为3、6、15d.确定Q26和G18为最佳酸牛乳生产用菌种.     

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as “Weissella ghanaensis,” was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named “Acetobacter senegalensis” (A. tropicalis-like) and “Acetobacter ghanaensis” (A. syzygii-like).     

乳糖酶水解牛乳的乳酸菌发酵研究

酵母乳糖酶水解牛乳经乳酸菌发酵时,其凝乳时间、产酸速度和ｐＨ下降均比普通乳为快。但对乳酸菌的生长影响不大。两种乳制作的酸奶,它们的组织状态、口感和香味物质乙醛也没有差异,但双乙酰和奶油香似乎略差些。     

Fermentation of Fructooligosaccharides by Lactic Acid Bacteria and Bifidobacteria

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.     

Fermentation of Aqueous Plant Seed Extracts by Lactic Acid Bacteria

The effects of lactic acid bacterial fermentation on chemical and physical changes in aqueous extracts of cowpea (Vigna unguiculata), peanut (Arachis hypogea), soybean (Glycine max), and sorghum (Sorghum vulgare) were studied. The bacteria investigated were Lactobacillus helveticus, L. delbrueckii, L. casei, L. bulgaricus, L. acidophilus, and Streptococcus thermophilus. Organisms were inoculated individually into all of the seed extracts; L. bulgaricus and S. thermophilus were also evaluated together as inocula for fermenting the legume extracts. During fermentation, bacterial population and changes in titratable acidity, pH, viscosity, and color were measured over a 72-h period at 37°C. Maximum bacterial populations, titratable acidity, pH, and viscosity varied depending upon the type of extract and bacterial strain. The maximum population of each organism was influenced by fermentable carbohydrates, which, in turn, influenced acid production and change in pH. Change in viscosity was correlated with the amount of protein and titratable acidity of products. Color was affected by pasteurization treatment and fermentation as well as the source of extract. In the extracts inoculated simultaneously with L. bulgaricus and S. thermophilus, a synergistic effect resulted in increased bacterial populations, titratable acidity, and viscosity, and decreased pH in all the legume extracts when compared to the extracts fermented with either of these organisms individually. Fermented extracts offer potential as substitutes for cultured dairy products.     

玉米秸秆青贮过程中优势细菌多样性分析

通过从发酵0、1、3、5、15、30和60d自然发酵的青贮玉米(CK)和采用菌剂处理的青贮玉米(处理1)上取样,分别进行pH值测定和菌群总DNA的提取。总DNA经纯化后采用引物341F-GC和518R进行细菌16S rDNA V3区PCR扩增,扩增产物进行变性梯度凝胶电泳(DGGE)分离并回收优势条带和变化明显的条带进行测序分析,了解秸秆饲料发酵过程中的优势细菌多样性及动态变化规律,从而为现有青贮接种菌的改良奠定理论基础。研究结果表明:采用菌剂处理的青贮玉米pH值下降更快,其优势细菌种类更丰富,且各优势菌的丰度比对照组中相应菌的丰度更强。     

Influence of Turning and Environmental Contamination on the Dynamics of Populations of Lactic Acid and Acetic Acid Bacteria Involved in Spontaneous Cocoa Bean Heap Fermentation in Ghana

The influence of turning and environmental contamination on six spontaneous cocoa bean heap fermentations performed in Ghana was studied through a multiphasic approach, encompassing both microbiological (culture-dependent and culture-independent techniques) and metabolite target analyses. A sensory analysis of chocolate made from the fermented, dried beans was performed as well. Only four clusters were found among the isolates of acetic acid bacteria (AAB) identified: Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter senegalensis, and a potential new Acetobacter lovaniensis-like species. Two main clusters were identified among the lactic acid bacteria (LAB) isolated, namely, Lactobacillus plantarum and Lactobacillus fermentum. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm and factory sites, indicating the cocoa pod surfaces and not the general environment as the main inoculum for spontaneous cocoa bean heap fermentation. Turning of the heaps enhanced aeration and increased the relative population size of AAB and the production of acetic acid. This in turn gave a more sour taste to chocolate made from these beans. Bitterness was reduced through losses of polyphenols and alkaloids upon fermentation and cocoa bean processing.     

Diversity of Lactic Acid Bacteria Associated with Fish and the Fish Farm Environment, Established by Amplified rRNA Gene Restriction Analysis

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus “faecium” group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.     

DNA分子标记技术在乳酸菌多态性研究中的应用

本文概述了常用于乳酸菌分离鉴定及多态性研究中的基于rDNA序列的分子标记技术和几种DNA指纹图谱技术(RAPD,ARDRA,AFLP,REP/ERIC-PCR),并对这些技术的原理、方法及其近几年在乳酸菌研究中的进展进行了介绍.同时本文还比较了各种方法的优缺点,不同的研究方法,其分辨率和检测限不同,必须根据研究目的,选择合适的分析方法.     

乳酸菌酸胁迫反应机制研究进展

乳酸菌可发酵糖类产生乳酸,并广泛应用于食品、药物和饲料等工业。由于有机酸的积累,乳酸菌大部分的生长代谢都在低pH的酸性环境中进行,具有酸胁迫反应。pH的自我平衡、ATR反应机制、对大分子的保护和修复作用及细胞膜的变化等是乳酸菌酸胁迫反应的主要机制,其中,pH自我平衡包括F0F1-ATPase质子泵、精氨酸脱氨酶途径(ADI)和谷氨酸脱羧酶途径(GAD)等。由此可见,乳酸菌酸胁迫反应机制涉及到基因和蛋白的表达调控等,是非常复杂的网络调控体系。     

Studies on Lactic Acid Fermentation

Resting cells of l. fermentum convert glyceraldehyde to equimolar lactic acid and neither the evolution of carbon dioxide nor the uptake of oxygen was observed. Glyceraldehyde-3-phosphate and 3-phosphoglycerate were identified as intermediates which were equally labeled with inorganic P³² in reaction systems, and the presence of triokinase was suggested.     

Antagonism Between Osmophilic Lactic Acid Bacteria and Yeasts in Brine Fermentation of Soy Sauce

Brine fermentation by osmophilic lactic acid bacteria and yeasts for long periods of time is essential to produce a good quality of shoyu (Japanese fermented soy sauce). It is well known that lactic acid fermentation by osmophilic lactic acid bacteria results in the depression of alcoholic fermentation by osmophilic yeasts, but the nature of the interaction between osmophilic lactic acid bacteria and yeasts in brine fermentation of shoyu has not been revealed. The inhibitory effect of osmophilic lactic acid bacteria on the growth of osmophilic yeasts was investigated. It was recognized that osmophilic shoyu yeasts such as Saccharomyces rouxii and Torulopsis versatilis were inhibited by a metabolite produced by osmophilic lactic acid bacteria (belonging to Pediococcus halophilus) in brine fermentation of shoyu. The primary inhibitor was considered to be acetic acid, although lactic acid was slightly inhibitory.     

Lactic Acid Bacteria for Mink

A method was developed to follow a lactic acid bacterial strain, Enterococcus faecium Cernelle 68, with respect to adhesion, multiplication, colonization, and persistence in the digestive tract of mink. Also the spread of the strain in the cage was examined. When adding 5 × 109 c.f.u. of a rifampicin resistant mutant per kg feed, high viable counts were registered throughout the digestive tract, apart from the oesophagus. Counts were increasing in the aboral direction, suggesting some multiplication in the intestine. It was possible to detect the strain in the intestinal tract 4 days after discontinuation of administration. Neither culture nor scanning electron microscopy gave evidence to suggest that E. faecium Cernelle 68 adhered to the mucosa. The spread of the E. faecium strain was observed in the environment. Counts of E. coli, lactobacilli, staphylococci, and Clostridia were low, and none of these bacteria were constant findings.     

Antagonism of Lactic Acid Bacteria against Phytopathogenic Bacteria

A variety of lactic acid bacteria, isolated from plant surfaces and plant-associated products, were found to be antagonistic to test strains of the phytopathogens Xanthomonas campestris, Erwinia carotovora, and Pseudomonas syringae. Effective “in vitro” inhibition was found both on agar plates and in broth cultures. In pot trials, treatment of bean plants with a Lactobacillus plantarum strain before inoculation with P. syringae caused a significant reduction of the disease incidence.     

Evolution of Yeasts and Lactic Acid Bacteria During Fermentation and Storage of Bordeaux Wines

The levels of yeasts and lactic acid bacteria that naturally developed during the vinification of two red and two white Bordeaux wines were quantitatively examined. Yeasts of the genera Rhodotorula, Pichia, Candida, and Metschnikowia occurred at low levels in freshly extracted grape musts but died off as soon as fermentation commenced. Kloeckera apiculata (Hanseniaspora uvarum), Torulopsis stellata, and Saccharomyces cerevisiae, the dominant yeasts in musts, proliferated to conduct alcoholic fermentation. K. apiculata and eventually T. stellata died off as fermentation progressed, leaving S. cerevisiae as the dominant yeast until the termination of fermentation by the addition of sulfur dioxide. At least two different strains of S. cerevisiae were involved in the fermentation of one of the red wines. Low levels of lactic acid bacteria (Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus spp.) were present in grape musts but died off during alcoholic fermentation. The malolactic fermentation developed in both red wines soon after alcoholic fermentation and correlated with the vigorous growth of at least three different strains of Leuconostoc oenos.     

Antioxidant Properties of Lactic Acid Bacteria

Microbiology - Lactic acid bacteria (LAB) are widely used in fermentation processes for the preparation of various foodstuffs, including dairy, meat and vegetable products. In the course of...